Detection of a single bacterial cell using a 16S ribosomal RNA-specific oligonucleotide probe designed to investigate periodontal pathogens

The current detection methods for periodontopathogens mainly use polymerase chain reactions. However, there are few methods available for visualizing the bacteria that impact on patients with periodontal disease for use in health education. The purpose of this study was to develop a specific detection method to visualize periodontopathogenic bacteria. Fluorescently-labeled oligonucleotide probes directed to specific 16S ribosomal RNA (rRNA) sequences of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were synthesized. Cultured individual bacterial species were fixed with 4% paraformaldehyde and smeared on glass slides. Fluorescein isothiocyanate-labeled oligonucleotide probes were hybridized under stringent conditions with smeared whole cells, and then probe specificity was investigated by epifluorescence microscopy. Comparatively long (50-mer) oligonucleotide probes for P. gingivalis and A. actinomycetemcomitans were designed. These probes clearly hybridized with 16S rRNA of the target species in situ and single bacterial cells were detectable visually. The probes exhibited no cross-hybridization against the additional organisms that were closely related to the target species. The fluorescence in situ hybridization technique is a specific and reliable method by which to visually identify the target organisms. The oligonucleotide probes designed in this study will be useful for detecting P. gingivalis and A. actinomycetemcomitans populations.